John S. Poulton, PhD

In autoimmune diseases, such as ANCA vasculitis, the targets of autoantibodies are one’s own proteins (autoantigens). The portion of an autoantigen recognized by an autoantibody is referred to as an epitope. Experimental data and immunological theory suggest that differences in epitope specificity can contribute to many aspects of autoimmunity, including disease initiation, clinical presentation, and response to treatment. Consequently, reactivity to certain epitopes can serve as biomarkers of disease activity and help guide the development of targeted therapeutic approaches.

Principal Components Analysis of epitope data from ANCA patients and healthy individuals suggests distinct epitope signatures can distinguish these groups.

To test key hypotheses in the field, we are performing epitope mapping of patient serum and plasma samples from our vast ANCA biorepository. Some of the questions we are answering include:

• Do epitope profiles change as a patient transitions from active disease to remission?
• Does epitope specificity evolve over the course of disease (i.e., epitope spreading)?
• Do the biophysical characteristics of epitopes change?
• Do epitopes found on different ANCA autoantigens indicate potential cross-reactivity?
• What can a patient’s epitope profile tell us about the potential cause of their disease (e.g., molecular mimicry following pathogen infection or drug-induced disease)?

Related publications:

Xi G, Mclnnis EA, Lardinois O, Hu P, Poulton JS, Free ME, Chen DP, Zeitler EM, Wu EY, Orzechowski NM, Derebail VK, Jennette JC, Falk RJ. Sequential carbonyl derivatives and hydrazone adduct formation on myeloperoxidase contribute to development of ANCA vasculitis. J Clin Invest. 2025 Feb 27;135(8):e178813. doi: 10.1172/JCI178813. PMID: 40020049; PMCID: PMC11996859.

Chen D, Moon Y-H, Peck BA, Taylor JJ, Gomez-Martinez I, Woodcock MG, Vincent BG, Falk RJ, Poulton JS, Bunch DO. Differentiating naturally occurring and disease associated autoantibodies in MPO-ANCA vasculitis through immunogenomic and epitope comparisons. Frontiers in Med.-Rheumatology. 2026 Feb 20.

Epitope mapping is frequently performed using in vitro binding assays, such as synthetic peptide arrays or chimeric antigens. Given the extreme diversity of the antibody repertoire within and among people, epitope mapping of circulating antibodies is complex and rife with caveats and confounders. To create a robust and highly reproducible approach to epitope mapping in ANCA vasculitis, we developed a novel yeast surface display library containing thousands of peptides derived from the protein sequences of known ANCA autoantigens. The overlapping peptide design of this library allows epitope resolution down to the single amino acid level. While we are currently screening patient samples for epitope mapping, we continue to improve and expand our library design to maximize its signal-to-noise and utility.

Protocol schematic of epitope mapping by yeast surface display.

Related publications:

Poulton JS*, Lamba S, Free M, Xi G, McInnis E, Williams G, Kudlacek ST, Thieker D, Kuhlman B, Falk R. High-resolution epitope mapping of commercial antibodies to ANCA antigens by yeast surface display. J Immunol Methods. 2024 May;528:113654. PMID: 38432292  *corresponding author

In addition to our studies of autoimmunity, we are interested in helping unravel the complexities of kidney podocytes. These critical cells reside in the renal glomerulus and serve key roles in kidney function. Podocytes are frequently damaged in many forms of kidney disease and we, like many in our field, have sought to improve our understanding of the molecular mechanisms that govern their unique architecture and function. Our podocyte studies utilize classic cell biological approaches in model organisms and cultured human podocytes.

Live cell microscopy and quantitative image analysis enables in vivo measurements of nephrin trafficking dynamics at the slit diaphragm.

Using Drosophila nephrocytes as a model of podocytes, we previously described a novel role for the apicobasal polarity machinery in formation of slit diaphragms via regulating the endocytic trafficking of slit diaphragm proteins, such as nephrin. We also demonstrated that disruption of endocytic trafficking triggers oxidative stress that further contributes to slit diaphragm defects.

Related publications:

Xi G, Lamba SA, Mysh, M, Poulton JS. Oxidative Stress Contributes to Slit Diaphragm Defects Caused by Disruption of Endocytosis. Kidney International Reports. 2024 Feb 9(2). PMID: 38344712

Mysh M, Poulton JS. The Basolateral Polarity Module Promotes Slit Diaphragm Formation in Drosophila Nephrocytes, a Model of Vertebrate Podocytes. J Am Soc Nephrol. 2021 Jun 1;32(6):1409-142. Epub 2021 Apr 1. PMID: 33795424

We greatly appreciate our opportunities to collaborate with our colleagues on previous and ongoing studies of podocyte biology:

Exploring tumor-gut-kidney interactions with Dr. Wu-Min Deng at Tulane University

• Cong F, Bao H, Wang X, Tang Y, Bao Y, Poulton JS, Liu X, Wong AC, Ji X, Deng WM. Translocation of gut bacteria promotes tumor-associated mortality by inducing immune-activated renal damage. EMBO J. 2025 Jul;44(13):3586-3613. PMC12217037.

Characterizing the dynamics and mechanisms of slit diaphragm protein trafficking with Dr. Tobias Hermle at University Medical Center Freiburg, Germany

• Lang K, Milosavljevic J, Heinkele H, Chen M, Gerstner L, Spitz D, Kayser S, Helmstädter M, Walz G, Köttgen M, Spracklen A, Poulton JS, Hermle T. Selective endocytosis controls slit diaphragm maintenance and dynamics in Drosophila nephrocytes. eLife. 2022 Jul 25;11. PMID: 35876643

John Poulton’s Bibliography